The long-term goat of this proposal is to carry out X-ray crystallographic studies on Hsp40 to uncover the basic mechanisms by which Hsp40 interacts with non-native polypeptides and cooperates with Hsp70 to perform the molecular chaperone activity. We have proposed an "anchoring and docking" model to illustrate the mechanisms for Hsp40 to interact with HspT0. To support this hypothesis, we intend to determine the crystal structure of the yeast type II Hsp40 Sis1 complexed with yeast Hsp70 Ssa1. We have constituted and crystallized the protein complex of Sis1 peptide-binding fragment and the Ssal C-terminal domain. Once solved, the complex crystal structure of Sis1 and Ssa1 will inform us how Hsp40 interacts with Hsp70 to carry out the non-native polypeptide delivery and assists Hsp in protein folding. The crystal structure of yeast type I Hsp40 Ydjl, complexed with the peptide substrate GWLYEIS, indicates that the pocket on the molecular surface of Ydjl may play important roles in peptide substrate binding. This pocket may exhibit significant plasticity to accommodate various sizes of hydrophobic side chains. We propose to determine the crystal structures of Ydjl peptide-binding fragment, complexed with peptide substrates GWWYEIS, and GWAYEIS. The crystal structures of Ydj1 and various peptide substrates may provide the structural basis of how Hsp40 adjusts itself to accommodate different peptide substrates. To reveal the mechanism by which Hsp40 primes the non-native polypeptide for the subsequent Hsp70 binding, we have designed a non-native polypeptide, Y1, by connecting two Hsp40 Ydj1 peptide substrates GWLYEIS with a flexible linker. The recombinant polypeptide Y1 showed a reasonable binding affinity to Hsp40 Ydjl. We intend to crystallize and determine the structure of the Ydjl complexed with the "non-native polypeptide", Y1, to illustrate how Hsp40 may affect the conformation of the non-native polypeptide. Finally, we will conduct structure-based mutagenesis studies to test our proposed models for the mechanisms of Hsp40 chaperone actions.